The most fundamental process in all of biology is the encounter of two or more molecules to form a specific complex. It is critical that such binding events occur within a time frame that is dictated by the survival needs of the organism. In the case of genomic DNA damage, extremely rare damaged bases must be located and removed by enzymes in a time constraint dictated by the next DNA replication event; otherwise deleterious mutations will be permanently fixed in the genome. Remarkable examples of highly efficient damage recognition are found with DNA base excision repair (BER) glycosylases. These enzymes locate and cleave the glycosidic bond of rare damaged bases in DNA by tracking along the DNA chain, beginning the BER cascade. The goal of this proposal is to develop defined in vitro model systems of increasing complexity to dissect the fundamental solution properties that influence DNA chain tracking by human uracil DNA glycosylase (hUNG). We will then evaluate the mechanism of facilitated diffusion in the complex crowded environment of human cells. These comprehensive studies will uncover general principles for search and recognition and will test these principles by engineering enzymes with enhanced DNA tracking properties. The first Specific Aim will extend our new 'molecular clock' approach to probe how DNA chain tracking is affected by crowded solution environments that mimic the cell nucleus. We will measure the efficiency of tracking between uracil sites embedded in a single DNA chain in the presence of uncharged crowding agents and also crowded solutions that contain high densities of nonspecific DNA. Exciting preliminary findings show that crowding greatly enhances DNA sliding. These studies will provide an essential mechanistic framework for interpreting transfer measurements in the crowded environment of living cells (Aim 3). The Second Aim will evaluate the role of electrostatics in DNA chain tracking. It is widely assumed that nonspecific electrostatic interactions allow proteins to track along nonspecific DNA but this has never been directly tested. We will dissect how these interactions affect DNA tracking by appending hUNG with short, positively charged peptide tails derived from naturally evolved DNA binding proteins. Specifically, these studies will determine how nonspecific electrostatic interactions change the residence time on nonspecific DNA, the 1D diffusion constant for sliding, the probability of hopping, and the overall efficiency of damage repair in dilute and crowded environments. The Third Aim evaluates the mechanism for locating U/A pairs in a human cell nucleus. We have developed two innovative strategies that allow investigation of facilitated diffusion and uracil recognition in human cells and the probability that hUNG will reac with one or two uracil sites placed on the same DNA chain as a function of site spacing. We have also developed the methodology to incorporate U/A base pairs into specific DNA sequences within the host genome. This provides a unique model system for following repair of U/A sites in genomic DNA using an innovative new sequencing platform (Uracil BE-Seq).